Victor x4 multi plate reader

Medium was removed by aspiration and 100 μl of room temperature Bright-Glo™ Luciferase Assay System (Promega) was added to the cells. After 5 minutes, the cells were lysed and the lysate was transferred to a black Costar EIA/RIA polystyrene half area 96-well plate (Corning). Luminescence was measured with the VICTOR™ X4 Multi-Plate Reader (PerkinElmer, Waltham, MA).

FcγR interactions with IgGs were measured by the dose-dependent upregulation of luciferase by Jurkat NFAT-luciferase (JNL) cells expressing FcγRs in response to antibody-opsonized target cells over a range of IgG concentrations. FcγR-transduced JNL cells (2×10⁵) were co-incubated with Raji cells (1×10⁵) in the presence of serial dilutions of anti-CD20 IgG antibodies. JNL cells were incubated overnight or for 4 hours with Raji cells in triplicate wells at each antibody concentration (200 μl/well final volume) in opaque white, flat-bottom, 96-well plates (Corning). At the end of the incubation, 50 μl BriteLite Plus luciferase substrate (PerkinElmer) was added to each well and luminescence was measured using a Victor X4 multiplate reader (PerkinElmer). Ramos cells (ATCC), which do not express FcγR2B, were also tested for antibody-mediated recognition by FcγR-transduced JNL cells. The different IgG subclasses and Fc domain variants of human and rhesus macaque IgG were run in parallel for each experiment.

KIR-CD28-CD3ζ⁺ JNL cells were incubated overnight with MHC class I-expressing 721.221 cells, or MHC class I-negative parental 721.221 cells, in R10 medium without G418, hygromycin or puromycin at 1:1 effector to target ratios (1×10⁵ JNL cells and 1×10⁵ 721.221 cells in 100 μl R10 medium) in triplicate wells of white 96-well plates. For blocking experiments, 721.221 cells expressing Mamu-AG were pre-incubated with a mouse monoclonal antibody to Mamu-AG (clone 25D3) (43 (link)) or an isotype control antibody (clone 11513) for 1 hour at 37° C before the addition KIR3DL05–28Z JNL cells. After an overnight incubation, 100 μl of BriteLite Plus luciferase substrate (PerkinElmer) was added to each well, and luciferase activity was measured approximately 2 minutes later using a Victor X4 multiplate reader (PerkinElmer). The fold-induction of luciferase activity was calculated by dividing the mean relative light units (RLU) of luciferase upregulation by the KIR-28Z JNL cells incubated with MHC class I-transduced 721.221 cells by the mean RLU for KIR-28Z JNL cells incubated with MHC class I-deficient parental 721.221 cells.